Carbonic anhydrase (carbonic dehydratase) is an enzyme widely occurring in animals and plants, which catalyzes the following reaction. EQU CO.sub.2 +H.sub.2 O.rarw..fwdarw.H.sup.+ +HCO.sub.3.sup.-
In C.sub.3 plants, it is thought that carbonic anhydrase plays a role in preventing evaporation of CO.sub.2 from chloroplasts by converting CO.sub.2 to carbonate ion. One of substrates of ribulose bisphosphate carboxylase (Rubisco) which is an enzyme for carbon dioxide fixation is CO.sub.2. Thus, it is thought that carbonic anhydrase supplies the substrate of Rubisco. Localization of carbonic anhydrase in cells of higher plants varies depending on the type of photosynthesis of the plant. In C.sub.3 plants, carbonic anhydrase activity is found in chloroplasts and in C.sub.4 plants, carbonic anhydrase activity is mainly found in cytoplasm of mesophyll cells.
As mentioned above, carbonic anhydrase catalyzes the reaction by which equilibrium between CO.sub.2 and hydrogen carbonate ion (HCO.sub.3.sup.-) in a solution is maintained. Although this equilibrium is reached under natural conditions, it takes a long time to reach the equilibrium if the enzyme does not participate. Therefore, if this enzyme is introduced by genetic engineering technique into a C.sub.3 plant in which the enzyme is not localized in cytoplasm, it is thought that the reaction to reach the equilibrium between CO.sub.2 and HCO.sub.3.sup.- is promoted and so the substrate of the enzyme carrying out carbon dioxide fixation is efficiently supplied, so that the ability to carry out carbon dioxide fixation of the plant is promoted.
Recently, it was reported that phosphoenol pyruvate carboxylase (PEPC) which is an enzyme catalyzing the first carbon dioxide fixation reaction of C.sub.4 plants was introduced into a C.sub.3 plant by genetic engineering technique (Hudspeth, R. L. et al., (1992), Plant Physiol. 98:485-464; Katsura IZUI et al., (1993), Plant Cell Technology 5: 74-82). The substrate of this enzyme is HCO.sub.3.sup.-. Since carbonic anhydrase does not exist in cytoplasm of C.sub.3 plants, in order that PEPC expressed in the cytoplasm efficiently functions, it is necessary to sufficiently supply HCO.sub.3.sup.-. Thus, if carbonic anhydrase is introduced into the plant to which PEPC has been introduced, it is thought that HCO.sub.3.sup.- consumed by the enzyme reaction of PEPC is supplied to cytoplasm, so that the ability of carbon dioxide fixation of the plant can be further promoted.
Carbonic anhydrase genes of dicotyledons such as spinach (Burnell, J. N. et al., Plant Physiol 92:37-40 (1990); Fawcett, T. W. et al., J. Biol. Chem. 265:5414-5417), pea (Roeske, C. A. et al., Nucleic Acid Res. 18:3413 (1990); Majeau, N. et al., Plant Physiol. 95:264-268 (1991)), Arabidopsis thaliama (Raines, C. A. et al., Plant Mol. Biol. 20:1143-1148 (1992)) and tobacco (Majeau, N. et al., EMBL Nucleotide Sequence Databases, Accession No. M94135, 1992)) have been isolated and sequenced. However, since the carbonic anhydrases of monocotyledons have enzyme properties different from those of dicotyledons, it is expected that greater effects will be obtained by introducing a carbonic anhydrase gene of a monocotyledon to monocotyledons.
As for carbonic anhydrase genes of monocotyledons, maize carbonic anhydrase gene has been partially sequenced (Keith et al., Plant Physiol. 101:329-332 (1993)). However, the sequenced region is only 210 bp. It is thought that this is too short to encode an active carbonic anhydrase and so cannot be used for genetic manipulation of monocotyledons.